Self-immolative nanocapsules precisely regulate depressive neuronal microenvironment for synergistic antidepression therapy

Background Pharmacotherapy constitutes the first-line treatment for depression. However, its clinical use is hindered by several limitations, such as time lag, side effects, and narrow therapeutic windows. Nanotechnology can be employed to shorten the onset time by ensuring permeation across the blood brain barrier (BBB) to precisely deliver more therapeutic agents; unfortunately, formidable challenges owing to the intrinsic shortcomings of commercial drugs remain. Results Based on the extraordinary capability of monoamines to regulate the neuronal environment, we engineer a network nanocapsule for delivering serotonin (5-hydroxytryptamine, 5-HT) and catalase (CAT) to the brain parenchyma for synergistic antidepression therapy. The nanoantidepressants are fabricated by the formation of 5-HT polymerization and simultaneous payload CAT, following by surface modifications using human serum albumin and rabies virus glycoprotein. The virus-inspired nanocapsules benefit from the surface-modifying strategies and exhibit pronounced BBB penetration. Once nanocapsules reach the brain parenchyma, the mildly acidic conditions trigger the release of 5-HT from the sacrificial nanocapsule. Releasing 5-HT further positively regulate moods, relieving depressive symptoms. Meanwhile, cargo CAT alleviates neuroinflammation and enhances therapeutic efficacy of 5-HT. Conclusion Altogether, the results offer detailed information encouraging the rational designing of nanoantidepressants and highlighting the potential of nanotechnology in mental health disorder therapies. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1186/s12951-023-02008-9.

of each sample according to the standard curve.

Structure analysis of poly(5-HT), degraded poly(5-HT), and 5-HT
To comprehensively investigate the polymerization of 5-HT in this study, we synthesized poly(5-HT) using the same method without the presence of proteins. In brief, 0.45 mL of the aqueous solution of 5-HT (C5-HT = 32 mg/mL) was added to 21 mL of Tris-HCl buffer (pH 9.5) and vigorously stirred at room temperature for 18 h. Furthermore, 0.6 mL of the 5-HT aqueous solution was added to the reaction mixture and vigorously stirred for 2 h at room temperature. The resulting mixture was then centrifuged at 1000 rpm for 10 min, and the supernatant was retained and purified by membrane dialysis (2 kDa) in ultrapure water at pH 9.5 for 6 h. The product was obtained through lyophilization. Subsequently, the powder of poly(5-HT) was incubated with a pH 6.4 buffer (Citric acid-Na2HPO4 buffer) for 72 h. The mixture was then centrifuged using an ultracentrifuged tube (with a cutoff molecular weight of 3 kDa) at 8000 rpm for 30 min. The eluent was collected, and the degraded poly(5-HT) was obtained through lyophilization. The structure of poly(5-HT), degraded poly(5-HT), and 5-HT was measured using FTIR (Nicolet is 50, Thermo Scientific, USA), X-Ray Photoelectron Spectroscopy (XPS, ESCALAB Xi+ , ThermoFischer, USA), Electrospray Ionization Mass Spectroscopy (ESI-MS, LTQ Orbitrap Velos Pro, Thermo Scientific, USA). For XPS analysis, the spectrometer employed an AI Kα X-ray source (hv=1486.6eV) under ultrahigh vacuum conditions (8×10 -10 Pa). Spectra were acquired with a pass energy of 100 eV, using 0.05 eV steps for the survey spectra and 30 eV for high-resolution spectra of C 1s, O 1s, and N 1s. In addition, the poly(5-HT) sample was centrifuged at 1000 rpm for 10 minutes, and the supernatant was collected for ESI-MS analysis. spectrum and fluorescence spectrum at 678 nm excitation and 788 nm excitation, respectively (Fig. S26 and Fig. S36). Next, 866 μL of FITC/DMF solution at a concentration CFITC = 1 mg/mL (445 μM) and 15 mg of RVG29 (890 μM) were dissolved in 5mL of pH 9.5 buffer (Tris-HCl buffer). The reaction mixture was allowed to stir for 5 h at RT. The unlabeled FITC was removed by membrane dialysis with a 2 kDa cutoff for 6 h. The final product, FITC-labeled RVG29 (RVG29-FITC), was dried through lyophilization. Then, we used HSA-Cy5.5 and RVG29-FITC to synthesize double-labeled VCNCs and VNCs by the method described above. The FITC labeling efficiency was also tested by the UV-vis spectrum and fluorescence spectrum (Fig. S30).

Cellular anti-inflammation by incubating with nanocapsules and reagents
PC-12 cells were seeded into 24-well plates (surface area/well = 1.9 cm 2 ) at a density of 150,000 cells/well with a volume of 1 mL and were incubated overnight. Then, the cells were respectively exposed to VCNCs, CNCs, VNCs, 5-HT, CAT, and HSA at different concentrations and incubated for 3 h. The specific concentrations of the samples used for the study are provided in Table S5. After exposure, the cell medium was removed, and the cells were washed twice with 1 mL of PBS. The cells were then incubated with 1.0 mL FBS-supplemented medium containing 1.0 μL ROSUP reagent for 0.5 h at 37°C. As a reference control, one group of cells was not exposed to samples or the ROSUP reagent. We then used DCFH-DA to detect the level of ROS.
The data were recorded using the fluorescent microplate reader (Envision@2015, PerkinElmer, USA). Furthermore, the intracellular ROS level was visualized using the inverted fluorescence microscope (Eclipse Ti2, Nikon, Japan) at the FITC channel.

Behavioral tests
To establish SPT, we used the two-bottle free-choice method. Mice were individually housed during the measurement and trained to adapt to two bottles of 1.5% sucrose solution (w/v) in each cage for 2 h, 48 h prior to the test. Before 24 h, mice were trained to adapt to one bottle of 1.5% sucrose solution (w/v) and one bottle of ultrapure water for 2 h. Following this, mice were completely deprived of food and water, and presented with two pre-weighed bottles for 2 h, one containing 1.5% sucrose solution and the other containing pure water. The bottles were weighed again after the measurement, and the sucrose preference was calculated as a percentage using the following equation: Sucrose preference [%] = sucrose consumption / (sucrose consumption + water consumption) × 100. For OFT, each mouse was placed in a black open box (60 cm × 40 cm × 30 cm) within a quiet room. The testing session lasted 5 min, preceded by a 3-min habituation session. The total traveled distance was recorded and analyzed using Smart V03, while the percentage of the distance traveled in the center zone was used to measure locomotion in the open field. In FST, mice were placed individually in a plastic cylinder (30 cm height × 11 cm diameter) containing water at 25°C in a quiet room. The mice were allowed to adapt to the environment for 15 min before 24 h. During the measurements, the mouse was placed in the water and forced to swim for 5 min, and the immobility time within this period was recorded using the video tracking system. Smart V03 was used for the analysis of immobility time.

Western blot analysis
To detect protein expression, the hippocampus samples were homogenized in RIPA buffer containing phosphatase inhibitors. The lysates were then centrifuged at 15,000g for 15 min at 4 °C to remove cellular debris. The resulting supernatants were diluted 20 times and subjected to the Bradford assay (Beyotime, #P0006C, China) for protein quantification. For protein separation, 40 μg of protein was loaded onto SDS-PAGE gels, with 8% polyacrylamide gel used for Nrf2 and 12% polyacrylamide gel used for BDNF. The gels were subsequently transferred onto PVDF membranes using standard procedures, with a transfer time of 120 min at 200 mA for Nrf2 and 40 min at 200 mA for BDNF. The PVDF membranes were then subjected to immunoblotting by incubating them with primary and secondary antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence (Beyotime, #P0018AS, China) and captured using a gel documentation system (Bio-Rad). The following antibody dilutions were used: 1:2000 for rabbit polyclonal antibodies against Nrf2 (Proteintech, #16396-1-AP, China), 1:1000 for rabbit monoclonal antibodies against BDNF (Abcam, #ab108319, UK), 1:2000 for rabbit polyclonal antibodies against β-actin (Proteintech, #120536-1-AP, China), and 1:2000 for goat anti-rabbit IgG (H+L) peroxidase/HRP-conjugated secondary antibodies (Elabscience, #E-AB-1003, China).

Slice staining
The ROS fluorescent staining was performed on frozen brain slides. The slides were left at room temperature for 30 min, then incubated with anti-fluorescence quenching agents for 5 min and washed with running water for 10 min. Next, the slides were stained with ROS fluorescence solution (Sigma, #D7008, United States) at 37°C for 30 min, followed by incubation with DAPI solution (Servicebio, #G1012, China) at room temperature for 10 min. The slides were then washed with PBS three times for 5 min each. Lastly, the slides were visualized using the fluorescent imaging system (Eclipse C1 and DS-U3, Nikon, Japan).
For immunofluorescence staining, the frozen slides were allowed to reach room temperature for 30 min. Subsequently, the slides were incubated with PBS for 10 min.
Following this, permeabilization agent (Proteintech, #PR30007, China) was applied to the slides for 20 min, followed by block solution (Proteintech, #PR30008, China) for 1 h. Next, the slides were incubated overnight at 4 °C with the primary antibodies.
The dilutions of the primary antibodies used were as follows: 1:200 for goat polyclonal antibodies against Iba 1 (Abcam, #ab5076, UK) and 1:500 for rabbit polyclonal antibodies against GFAP (Abcam, #ab7260, UK). After incubation, the slides were washed three times with PBS for 10 min each. For the subsequent step, the slides were incubated with the secondary antibodies for 1 h at 37 ℃. The dilutions of the secondary antibodies used were as follows: 1:500 for donkey anti-goat IgG H&L (Alexa Fluor® 488) (Abcam, #ab150129, UK) and 1:250 for donkey anti-rabbit IgG(H+L) (CoraLite594) (Proteintech, #SA00013-8, China). After incubation, the slides were washed three times with PBS for 10 min each. Finally, the slides were mounted with an anti-fading agent containing DAPI (Solarbio, #S2110, China).
For Hematoxylin-eosin staining (HE) staining, the first step is the dewax procedure.
The organ slides were placed at 60°C for 2 h, followed by deparaffinization with xylene (20 min for three times) and alcohol solutions with gradient concentrations (5 min in 100% ethanol twice, 5 min in 90%, 80%, and 70% ethanol) and rehydration in ultra-pure water (5 min). The slides were then treated with PBS three times for 5 min.
Afterward, the slides were stained with hematoxylin for 10 min, washed with running water for 1 min, differentiated with 1% hydrochloric acid ethanol for 30 seconds, washed with running water for 1 min, placed with blue returning liquid (Servicebio, #G1040, China) for 30 seconds, and washed with running water for 1 min. Thereafter, the slides were stained with eosin (Biosharp, #BL700A-2, China) solution for 2 min.
The final step is dehydration, where the tissue slides were immersed in gradient concentrations of ethanol (5 min in 100% ethanol twice, 5 min in 90%, 80%, and 70% ethanol) and fresh xylene (10 min). Finally, the slides were sealed with neutral gum. For brain slides, the samples were visualized by Vectra Polaris (Akoya Bioscience, USA), while for major tissue slides, the samples were captured by a microscope (Zeiss, Germany).
For immunohistochemistry (IHC) staining, the dewax procedures were conducted after obtaining the paraffin slides, which are described in HE staining. The slides were microwaved in EDTA solution (pH = 9) for 20 min to repair antigens, washed with ultra-pure water three times for 5 min, and incubated with 3% hydrogen peroxide solution for 10 min. Next, slices were washed with ultra-pure water twice for 5 min, PBS once for 5 min, and blocked with 10% goat serum (Solarbio, #SL038, China) for 1 h at room temperature. Then, the slides were covered with primary antibodies, including anti-BDNF (Abcam, #ab108319, UK, 1:200) and 5-HT (Serotonin) Rabbit Antibody (Immunostar, #20080, USA, 1:2000), and incubated at 4°C overnight. On the next day, the slides were put at 37°C for 30 min, removed the primary antibodies, covered with the secondary antibodies (1:400, Goat Anti-Rabbit IgG (H+L) (peroxidase/HRP conjugated, Elabscience, China), and incubated for 30 min at 37°C.
Then, the slides were washed with PBS three times for 5 min, stained with a DAB kit (ZSGB-BIO, China), and washed with running water for 3 min. Finally, the slides were stained with hematoxylin and dehydrated with alcohol and xylene, following the protocols described in HE staining.
For Nissl staining, the slides were first dewaxed using the procedures described in HE staining. Subsequently, the slides were stained with the Nissl kit for 10 min at room temperature, followed by two washes with ultrapure water for 5 min each.
Lastly, the slides were dehydrated using the methods described in HE staining.    The labeling efficiency of α-CT-FITC has a significant difference between the theoretical result.
α-CT easily precipitate in pH 9.5 Tris-HCl buffer, which is close to its isoelectric points (8.9).       correlation between the peak area from HPLC spectra and 5-HT concentration was used to construct a standard curve.          Figure S11.  Consequently, the resulting nanocapsules demonstrated improved solubility characteristics.      Table S4). PC-12 cells were incubated with test samples in the cell medium for 24 h and 48 h. Cell viability was assessed using the resazurin assay and presented as the mean ± standard deviation of n≥3 measurements.  Table S4). bEnd.3 cells were incubated with test samples in the cell medium for 24 h and 48 h. Cell viability was assessed using the resazurin assay and presented as the mean ± standard deviation of n≥3 measurements.  bEnd.3 cells which had been exposed to VCNCs-Cy5.5, CNCs-Cy5.5, and VNCs-Cy5.5 at a concentration C5-HT = 25 μg/mL for 1, 3, and 5 h. In the uptake studies, bEnd.3 cells were exposed to nanocapsules for a duration of texp = 3 h, resulting in a total cell count of Ncell (texp:3h). Subsequently, PC-12 cells were co-incubated with bEnd.3 cells for a further duration of tinc = 24 h, resulting in a total cell count of Ncell (texp:3h + tinc:24h). The number of cells had increased by a factor of Ncell (texp:3h + tinc:24h)/ Ncell (texp:3h) from the initial cell count at texp:3h. h. The absolute cell numbers at texp = 3 h were 111.4±7.8 ×10 4 cells/mL (VCNCs) and 119.7 ± 18.8×10 4 cells/mL (CNCs). The factor Ncell(texp:3h + tinc:24h)/ Ncell(texp:3h) was calculated accordingly. and VNCs-Cy5.5+FITC were also analyzed.    VNCs, 5-HT, CAT, and HSA at different concentrations. The legend "triangle" refers to samples exposed at higher concentrations to lower concentrations as indicated in Table S5. Fluorescence was detected by the microplate reader. Results are from three independent experiments. PC-12 cells incubated with DCFH-DA were used as the blank group. The significant differences between groups were analyzed by one-way ANOVA method, * *P＜0.01, * * * P＜0.001.  Table S5. The intensity of Fluo-4 fluorescence is proportional to the level of cytoplasmic calcium. PC-12 cells incubated with Fluo-4AM were used as the blank group. Results are from three independent experiments. The significant differences between groups were analyzed by the one-way ANOVA method, *P＜0.05, **P＜0.01.     ELISA detection of hippocampal Nrf2 levels following treatments, with normalization to the protein concentration of each sample (n = 3). The significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05, **P < 0.01, *** P < 0.001.  by Image J. The significant differences between groups were analyzed using the one-way ANOVA method, *P < 0.05, **P < 0.01, *** P < 0.001.